Primary Pulmonary Hypertension (PPH) is a rare disease of the pulmonary vasculature, resulting in abnormally high pressure in the pulmonary artery, right ventricular failure, and death. While the pathogenesis of PPH is not clearly understood, the increased pulmonary vascular resistance observed is attributed to three main factors: 1) vasoconstriction, 2) thickening of the vessel wall due to vascular remodeling, and 3) in situ thrombosis. (Archer, S., Rich, S. Circulation 2000, 102, 2781-2791; Rich, S. Curr. Treat Options. Cardiovasc. Med. 2000, 2, 135-140). There is no cure for PPH and the treatment options are limited. (Naeije, R., Vachiery, J. L. Clin. Chest Med. 2001, 22, 517-527). The survival rate is less than a year in patients presented with New York Heart Association Functional Class IV symptoms. Although lung transplantation is a possible treatment option, there are obvious drawbacks such as shortage of donor organs and chronic rejection as well as the possibility of infection. (Franke, U., Wiebe, K., Harringer, W., Franke, T., Wittwer, T., Wahlers, T., Haverich, A. Eur. J. Cardiothorac. Surg. 2000, 18, 447-452). Long term intravenous treatment with epoprostenol (also known as prostacyclin or PGI2) has greatly increased the survival rate.
Prostacyclin or PGI2 (Prostaglandin I2) is a major cyclooxygenase metabolite of arachidonic acid, and is mainly produced by vascular endothelial cells. Prostacyclin is a prostaglandin, which occurs along with other eicosanoids like thromboxanes and leukotrienes. Prostacyclin is a potent vasodilator with platelet-inhibitory, anti-proliferative and fibrinolytic activities. (Moncada, S., Vane, J. R. Pharmacological Rev. 1979, 30).
The exact levels of prostacyclin in human plasma are unknown, however like most eicosanoids it occurs in very low amounts (pg/mL). Thromboxane B2 (metabolite of thromboxane A2, a vasoconstrictor) and its metabolites occur in very low amounts like 1-10 pg/mL in plasma. Leukotrienes generally function as vasoconstrictors and bronchoconstrictors and occur in varying amounts in plasma and urine. Leukotriene B4 occurs between 50 pg/mL-50 ng/mL, leukotriene E4 occurs between 80 pg/mL-1000 ng/mg of creatinine. Prostaglandin F2α concentrations are found between 10 pg/mL-100 pg/mL, prostaglandin D2 is generally found at 5 pg/mL-500 pg/mL, and prostaglandin F1α occurs at 1 ng/mL-100 ng/mL.
Currently intravenous epoprostenol is indicated for the chronic treatment of primary pulmonary hypertension (PPH) in New York Heart Association (NYHA) functional Class III and Class IV patients who do not respond to conventional therapy. (Krishnan, U. Indian J. Pediatr. 2000, 67, 523-527; Nauser, T. D., Stites, S. W. Am. Fam. Physician 2001, 63, 1789-1798). There is no set dose of prostacyclin for PPH and the starting dose varies between 4-8 ng/kg/min. Following the initiation of intravenous prostacyclin, most patients require an automatic dose increase of 2 ng/kg/min every two weeks until the rate of administration reaches 20 ng/kg/min. Further dose increase of 0.5 to 1 ng/kg/min per week is needed only upon the return of symptoms. Common side effects of intravenous prostacyclin include flushing, muscle pain, and abdominal cramps and these symptoms are not necessarily dose dependent. Thus, a method to quantify and correlate the plasma levels of prostacyclin with the onset of the untoward side effects or functional improvement would be useful in determining the optimal level of plasma prostacyclin for an individual patient.
A method to quantify the plasma levels of prostacyclin would be useful in determining the optimal level of plasma prostacyclin for an individual patient. Prostacyclin is very unstable with a half-life of only 60 minutes in plasma and 2-3 minutes in buffer. The immediate and stable product of hydrolysis of prostacyclin, 6-Keto-Prostaglandin F1α (6-keto-PGF1α), is quantifiable and can be used as a surrogate marker of the level of plasma prostacyclin. The detection method for prostacyclin or its breakdown product has to be extremely sensitive and accurate since either molecule is present in very low amounts (pg/mL) in the body. Moreover, the existing methods are time consuming mainly due to the necessity to extract 6-keto-PGF1α from biological samples. (Schramm, W., Smith, R. H., Jackson, T. M., Craig, P. A., Grates, H. E., and Minton, L. L. Clinical Chem. 1990, 36, 509-514; Tonai T., Y., K., Yano, T., Hayashi, Y., Yamamoto, S., Yamashita, K., and Miyazaki, H. Biochim. Biophys. Acta 1985, 836, 335-343). Thus there is a need for a rapid and sensitive detection method for prostacyclin. The present invention provides a single-step, solid phase immunoassay for 6-keto-PGF1α using the bioluminescent protein aequorin.